Prophylactic and therapeutic treatment of the ductal epithelium of a mammary gland for cancer

ABSTRACT

The present invention provides prophylactic and therapeutic methods of treating the ductal epithelium of an exocrine gland, in particular a mammary gland, for disease, in particular cancer. The methods comprise contacting the ductal epithelium of the exocrine gland with an epithelium-destroying gent, preferably by ductal cannulation, so as to realize a prophylactic or therapeutic effect.

This application claims priority to U.S. patent application Ser. No.08/510,623, which was filed on Aug. 3, 1995, and has been converted toU.S. provisional patent application Ser. No. not yet known.

The invention disclosed in this application was made with governmentsupport under NIH planning grant P20 CA/ES66205, “Gene-mediatedprevention of Cancer,” and grant NIH 1RO1 CA 57993, “Genetic andhormonal factors in mammary carcinogenesis,” awarded by the NationalInstitutes of Health, and with support of the American Cancer Societyunder grant RD388, “Targeted disruption of breast cells: a novelstrategy for cancer prevention.” Therefore, the government has certainrights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of an epithelium-destroyingagent to destroy the epithelium of an exocrine gland, particularly themammary gland, in the prophylactic and therapeutic treatment of disease,in particular cancer.

BACKGROUND OF THE INVENTION

Exocrine glands are glands that release a secretion external to or atthe surface of an organ by means of a canal or duct. Examples ofexocrine glands include, among others, the mammary glands, prostate,liver, gall bladder, pancreas, kidneys, sweat glands, and salivaryglands. Cancers of exocrine glands pose a major health problem,frequently resulting in death. Currently, cancers of the breast andprostate are among the leading causes of death among women and men,respectively.

The mature human breast comprises from six to nine major ducts, whichemanate from the nipple, serially branch into ducts and terminate inlobuloalveolar structures (Russo et al., Lab. Invest. 62(3): 244-278(1990)). This branching network of ducts is composed of epithelial cellsin a supporting matrix of connective tissue and endothelial cells.

Tissues removed from the human female breast during surgery and autopsyhave been examined in numerous studies directed to the nature and siteof origin of neoplastic growth. Subgross sampling and histologicalconfirmation have enabled pathological characterization of entirebreasts, leading to the postulation of the existence of four majorpossible sites of origin of mammary carcinomas, namely ducts, terminalducts, ductules, and acini (Russo et al., supra). Ductal origin issupported by the presence of more extensive epithelial proliferations,which are presumed to be preneoplastic, in surgically removed cancerousbreasts as compared to nonmalignant breasts removed during autopsies(Russo et al., supra).

With a cumulative lifetime risk of a woman developing breast cancerestimated to be 1 in 9, there is an urgent need to develop therapeuticmethods of treatment that are more effective, less invasive andaccompanied by fewer side effects and prophylactic methods of treatmentthat are more effective than increased and intensified physicalmonitoring and far less extreme than radical mastectomy. In spite of therecent discovery of the heritable breast cancer susceptibility loci,BRCA1 (Miki et al., Science 266: 66-71 (1994)) and BRCA2, and othercancer susceptibility loci, and the increasing ability of physicians toidentify women with elevated breast cancer risk, prophylactic methodsare still currently limited to physical monitoring and prophylacticmastectomy.

In view of the above, it is an object of the present invention toprovide a method of locally treating an exocrine gland prophylacticallyfor disease. It is another object of the present invention to provide amethod of locally treating an exocrine gland, in particular the mammarygland, prophylactically for cancer. Another object of the presentinvention is to provide a method of locally treating an exocrine glandtherapeutically for disease. Yet another object of the present inventionis to provide a method of locally treating a mammary glandtherapeutically for cancer. Still yet another object of the presentinvention is to provide a method of locally treating a mammary glandboth therapeutically and prophylactically for cancer. These and otherobjects and advantages, as well as additional inventive features, willbecome apparent from the detailed description provided herein.

SUMMARY OF THE INVENTION

The present invention provides prophylactic and therapeutic methods oftreating the ductal epithelium of an exocrine gland, in particular themammary gland, for disease, in particular cancer. The method comprisescontacting the ductal epithelium of the exocrine gland, in particularthe mammary gland, with an epithelial-destroying agent. The ductalepithelium is preferably contacted with the agent by ductal cannulation.The epithelium-destroying agent is preferably a vector comprising athymidine kinase gene, which is used in combination with ganciclovir(GCV), which can be systemically administered. Another preferredepithelium-destroying agent is a vector comprising a hypoxanthinephosphoribosyl transferase (HPRT) gene, which is used in combinationwith hypoxanthine aminopterin thymidine (HAT) nucleotide, which can besystemically administered. Also preferred as an epithelium-destroyingagent is a vector comprising a gene, which, upon transformation of acell of the ductal epithelium and expression therein, induces apoptosisor death of the transformed cell. A preferred apoptosis-inducing gene isbclxs. Other preferred epithelial-destroying agents include a cytolyticvirus, such as Vaccinia virus, and ethanol. The preceding methods canadditionally comprise contacting the ductal epithelium with a cytokineor hematopoietic growth factor, such as GM-CSF. Also provided by thepresent invention is a method of treating the ductal epithelium of amammary gland both therapeutically and prophylactically for cancer. Thecombined therapeutic/prophylactic method comprises treating the mammarygland therapeutically by surgery, radiation and/or chemotherapy andcontacting the ductal epithelium of the mammary gland, eitherconcomitantly or subsequently, with an epithelium-destroying agent,which does not specifically target cancerous cells. The combinedtherapeutic/prophylactic method can additionally comprise contacting theductal epithelium with a cytokine or hematopoietic growth factor, suchas GM-CSF.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the ratio of cell no. in the presence of GCV overcell no. in the absence of GCV (Cell No. +GCV/Cell No. −GCV) versusviral multiplicity of infection (viral MOI), which shows the effect ofthe addition of 10 μg/ml GCV on NMU68 and RBA rat tumor cell lines 6 hrsafter transduction with adenoviral-Herpes simplex thymidine kinase(AdHS-tk) at titers of 0, 100, 500 and 1,000 moi. After the cells weremaintained in the presence of GCV for 3 days, they were counted usingtrypan blue exclusion as a measure of cell viability and cell numberswere normalized to the growth of cells in the absence of ganciclovir(GCV).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the observation that the overwhelmingmajority of breast cancers arise from epithelial cells, particularlythose epithelial cells which line the ducts of the mammary gland and arecollectively referred to as the ductal epithelium. The present inventionis also based on the exocrine nature of the mammary gland. Given thatthe mammary gland is an exocrine gland, it was further observed that thecentral canal or duct could provide a means of directly accessing theductal epithelium for localized prophylactic and therapeutic treatmentof cancer. Based on these observations, the prophylactic and therapeuticmethods of the present invention were developed.

Prophylactic Method

The prophylactic method of the present invention is a method of treatingthe ductal epithelium of an exocrine gland prophylactically for adisease that affects the ductal epithelium of the exocrine gland. Themethod comprises contacting, preferably by ductal cannulation, theductal epithelium of the exocrine gland with an epithelium-destroyingagent so as to destroy cells of the ductal epithelium affected by thedisease.

In one embodiment of the prophylactic method of the present invention,the ductal epithelium of a mammary gland is treated prophylactically forcancer so as to inhibit the formation of cancer of ductal epithelialorigin. The method comprises contacting, preferably by ductalcannulation, the ductal epithelium of the mammary gland with anepithelium-destroying agent. The agent preferably is a vector comprisinga thymidine kinase gene, such as a Herpes simplex thymidine kinase gene,and ganciclovir, a vector comprising a HPRT gene and HAT nucleotide, acytolytic virus, such as a Vaccinia virus, or ethanol. The method canadditionally comprise contacting the ductal epithelium with a cytokineor hematcpoietic growth factor, such as GM-CSF.

In another embodiment of the prophylactic method of the presentinvention, the ductal epithelium of an exocrine gland, such as a mammarygland, is treated prophylactically for cancer so as to inhibit theformation of cancer of ductal epithelial origin. The method comprisescontacting, preferably by ductal cannulation, the ductal epithelium ofthe exocrine gland with an epithelium-destroying agent to destroy lessthan all of the ductal epithelium so as to inhibit the formation ofcancer of ductal epithelial origin. Preferably, up to about 70%, 80%,85%, 90% or 95% of the ductal epithelium is destroyed.

The epithelium-destroying agent is preferably a vector comprising athymidine kinase gene, such as that from Herpes simplex, andganciclovir, which can be brought into contact with the ductalepithelium by any suitable means, preferably by ductal-cannulation or bysystemic administration, a vector comprising a HPRT gene and HATnucleotide, which can be brought into contact with the ductal epitheliumby any suitable means, preferably by ductal cannulation or by systemicadministration, a vector comprising a gene, which upon transformation ofa cell of the ductal epithelium and expression therein, inducesapoptosis or death of the transformed cell, such as bclxs, ethanol, or acytolytic virus, such as Vaccinia virus.

The above method can additionally comprise the administration of acytokine or hematopoietic growth factor, such as GM-CSF. The GM-CSF canbe brought into contact with the ductal epithelium of the mammary glandby any suitable means, such as by ductal cannulation of GM-CSF or avector comprising a gene encoding GM-CSF, in which case the vector canbe the same vector as the one encoding the thymidine kinase, HPRT orapopotosis-inducing gene, or it can be systemically administered.

This embodiment of the prophylactic method can be used to treat anyexocrine gland. However, it is particularly useful in the treatment ofthe mammary gland.

The above-described prophylactic method of treating a mammary gland isparticularly useful in treating a mammary gland in a mammal at risk fordeveloping breast cancer. The mammary gland can be characterized as onethat has never had a tumor, one that had a tumor previously but thetumor is no longer detectable due to other prior therapeutic treatment,or one that has an incipient or occult tumor, preneoplasia or ductalhyperplasia. Normally, hyperplasias and incipient and occult tumors arenot detectable by means of physical examination or radiography.Accordingly, the prophylactic method will find use in cases where thereis reason to take some prophylactic measures, such as when there areknown inherited factors predisposing to cancers, where there aresuspicious lesions present in a breast with the potential for developinginto a malignancy, where there has been exposure to carcinogenic agentsin the environment, where age predisposes to a cancer, where cancer ofanother gland, e.g., the mammary gland of the contralateral breast,suggests a propensity for developing cancer, or where there is a fear orsuspicion of metastasis.

Therapeutic Method

The therapeutic method of the present invention is a method of treatingthe ductal epithelium of an exocrine gland therapeutically for a diseasethat affects the ductal epithelium of the exocrine gland. The methodcomprises contacting, preferably by ductal cannulation, the ductalepithelium of the exocrine gland with an epithelium-destroying agent soas to destroy cells of the ductal epithelium affected by the disease.

In one embodiment of the therapeutic method of the present invention,the ductal epithelium of a mammary gland is locally treatedtherapeutically for cancer so as to destroy cancerous and noncancerouscells of the ductal epithelium and inhibit the spread of cancer. Themethod comprises contacting, preferably by ductal cannulation, theductal epithelium of the mammary gland with an epithelium-destroyingagents which need not, and preferably does not, specifically targetcancerous cells. The agent preferably is a vector comprising a thymidinekinase gene, such as a Herpes simplex thymidine kinase gene, andganciclovir, a vector comprising a HPRT gene and HAT nucleotide, acytolytic virus, such as a Vaccinia virus, or ethanol. The method canadditionally comprise contacting the ductal epithelium with a cytokineor hematopoietic growth factor, such as GM-CSF.

In the therapeutic method, the epithelial-destroying agent shoulddestroy all of the diseased or malignant epithelium. In addition, theductal-epithelium immediately surrounding the diseased/malignantepithelium also preferably should be destroyed.

Combined Therapeutic/Prophylactic Method

The present invention also provides a method of treating the ductalepithelium of a mammary gland both therapeutically and prophylacticallyfor cancer. The method comprises treating the mammary glandtherapeutically with any given therapeutic method, such as thosecurrently known and used in the art. Examples of such methods includesurgical removal of the cancerous tissue, radiation therapy andchemotherapy. The method further comprises contacting, eitherconcomitantly with or subsequently to the therapeutic treatment, theductal epithelium of the mammary gland, e.g., by ductal cannulation,with an epithelium-destroying agent, which preferably does notspecifically target cancerous cells, so as to destroy any remainingcancerous cells and noncancerous cells and inhibit the spread of cancer.The epithelium-destroying agent is preferably a vector comprising athymidine kinase gene, such as a Herpes simplex thymidine kinase gene,combined with ganciclovir, a vector comprising a HPRT gene combined withHAT nucleotide, a cytolytic virus, such as a Vaccinia virus, or ethanol.The method can additionally comprise contacting the ductal epitheliumwith a cytokine or hematopoietic growth factor, such as GM-CSF.

Alternative Embodiments of Prophylactic & Therapeutic Methods

Although preferred embodiments have been described above, the methods ofthe present invention can be used to treat the ductal epithelium of anyexocrine gland. Examples of exocrine glands, other than the mammarygland, which can be treated with the present inventive methods include,among others, the prostate, liver, gall bladder, pancreas, kidneys,sweat glands, and salivary glands. The methods are especially useful inthe prophylactic and therapeutic treatment of the ductal epithelium ofmammary glands.

Similarly, the methods can be used to treat an exocrine gland for anydisease that affects the exocrine ductal epithelium. The methods areparticularly useful in the treatment of cancer, including the stages ofhyperplasia, adenoma, carcinoma in situ, and carcinoma, of ductalepithelial origin.

Any method can be used to destroy the ductal epithelium. It ispreferred, however, that the destruction is limited to the ductalepithelium or a part thereof.

Any method of contacting the ductal epithelium can be used to effectlocal treatment. Preferably, ductal cannulation is used. Although anyduct or lobule can be cannulated, it is preferred that the central canalor duct be cannulated. Ductal cannulation also enables direct injectionof a tumor mass, if desired.

Any epithelium-destroying agent can be used to destroy the ductalepithelium of an exocrine gland. The agent preferably should not destroycells other than cells of the ductal epithelium and preferably shouldnot result in side effects, the adversity of which outweigh the benefitsof destruction of the ductal epithelium. In no event should the methodsbe used to destroy completely the ductal epithelium of an exocrine glandin the prophylactic/therapeutic treatment of a given disease, whereinthe complete destruction of the ductal epithelium, in and of itself,would result in death of the mammal so treated.

Examples of agents that can be used in the context of the prophylacticand therapeutic methods of the present invention include cytotoxicagents. Any cytotoxic agent known in the art and suitable for contactingthe ductal epithelium of an exocrine gland of a mammal can be used. Inaddition to ethanol and GCV described above, other examples of cyotoxicagents and their prodrugs include genistein, okadaic acid_(,)1-β-D-arabinofuranosyl-cytosine, arabinofuranosyl-5-aza-cytosine,cisplatin, carboplatin, actinomycin D, asparaginase,bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil,cyclohexyl-chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin,etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogenmustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine,5-fluorouracil, 5-fluorocytosine, adriamycin, cyclophosphamide,methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogenagents such as tamoxifen, intracellular antibodies against oncogenes,the flavonol quercetin, Guan-mu-tong extract, retinoids such asfenretinide, nontoxid retinoid analogues such asN-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as limonene,perillyl alcohol and sobrerol. Preferably, the agent is locallyadministered, especially if administration of the agent is accompaniedby toxic side effects. Otherwise, the agent can be administered by anysuitable route, such as systemic administration.

A cytolytic virus also can be used as an agent. Any cytolytic virus canbe used as long as the organism mounts a rapid immunological response toit such that the virus cannot cause disease if it escapes the ductalepithelium. Examples of cytolytic viruses include Vaccinia viruses andSindbis viruses, which can also be used as vectors. Preferably, aVaccinia virus is used. Due to lack of mucosal immunity, Vacciniainfectious particles enter and lyse the breast epithelial cells, yetstromal immunity destroys the particles as soon as they leave theductile tree of the exocrine gland, thereby preventing cytolysis beyondthe ductal epithelium. The advantages of Vaccinia administration arethat it eliminates the need for high titer virus, the need to inducecell division in the breast, and the need to administer a drug to effectcell death.

A vector comprising a suicide gene also can be used as an agent, inconjunction with an agent that destroys the ductal epithelium of anexocrine gland. The vector comprising a suicide gene, upontransformation of a cell of the ductal epithelium and expressiontherein, renders the transformed cell sensitive to theepithelium-destroying agent, increases the sensitivity of thetransformed cell to the agent, converts the agent from a prodrug to anactive drug, activates the conversion of the agent from a prodrug to anactive drug, enhances the effect of the agent or, itself, produces aprotein that is cytotoxic. A preferred suicide gene for use in thepresent inventive methods is the one described above, i.e., a thymidinekinase, such as the one from Herpes simplex, which phosphorylates GCV,which, in turn, inhibits DNA replication. Another example of a suicidegene is cytosine deaminase, which is used in conjunction with5-fluorocytosine. If the vector comprising the suicide gene isadministered locally to the ducts, the cytotoxic agent or precursor canbe administered systemically, since only transfected cells will beaffected. In this regard, the bystander effect, i.e., the death ofneighboring uninfected cells, presumably due to transfer of toxicbyproducts through gap junctions between cells in the same compartment,obviates the need for every cell in the ductal epithelium, which is tobe destroyed, to be infected. However, sufficient time must be allowedbetween contacting the ductal epithelium with the suicide gene and theprodrug, for example, to achieve efficient killing of the breastepithelial cells.

A vector comprising an apoptosis-inducing gene also can be used as anagent that destroys the ductal epithelium of an exocrine gland (Vaux,Cell 76: 777-779 (1994)). Examples of apoptosis-inducing genes includeced genes, myc genes (overexpressed), the bclxs gene, the bax gene, andthe bak gene. The apoptosis-inducing gene causes death of transfectedcells, i.e., by inducing programmed cell death. For example, the bclxsgene, bax gene, or bak gene can be used to inhibit bcl-2 or bcl-x_(L′)leading to apoptosis. Where necessary, a vector comprising anapoptosis-inducing gene can be used in combination with an agent thatinactivates apoptosis inhibitors such as bcl-z, p35, IAP, NAIP, DAD1 andA20 proteins.

Suicide and apopotosis genes can be administered by way of a viralvector, such as an adenoviral or retroviral vector. Adenoviral vectorsenable the generation of high titer recombinant viruses (10¹¹/ml) andthe efficient transduction of postmitotic cells because adenoviral DNAexists as an episome in the nucleus (Verma, Molecular Medicine 1: 2-3(1994)).

According to one preferred embodiment, the gene can be under thetranscriptional regulation of a Rous sarcoma viral promoter.Alternatively, it can be under the control of an epithelialtissue-specific or cell-specific promoter.

Uptake of recombinant virus can be facilitated by pretreatment orsimultaneous treatment with polybrene or, for example, in the case of aretrovirus, attachment of the functional fragment of an antibody to theviral particle.

In another embodiment, the apoptosis gene or suicide gene can be presentin a recombinant microorganism, which will express the gene. Oneparticularly preferred microorganism for this purpose is the bacteriumListeria monocytogenes.

Other methods known in the art for introduction of raw DNA into cellscan be used in the methods of the present invention. Alternatively,liposomes, complexes between polypeptide ligands for receptors onmammary ductal epithelial cells, including complexes of antibodies andfunctional fragments thereof, and plasmids can be used (Mulligan,Science 260: 926-931 (1993)). Epithelial cell-specific promoters, suchas whey acidic protein (wap), can be used to target expression of agiven gene, e.g., a suicide gene, in ductal epithelial cells. Use canalso be made of wild-type tumor suppressor genes, such as p53 or Mcs-1(rat), homeobox genes expressed in normal cells but not in cancerouscells, and the maspin gene.

Additionally, the ductal epithelium dan be contacted with an agent toeffect the scavenging of epithelial cells destroyed in accordance withthe present invention, e.g., a cytokine/growth factor. Suitablecytokines/growth factors include GM-CSF, G-CSF, IL-2, IL-4, IL-6, IL-7,hCG, TNF-α, INF-α and INF-γ. Such factors can be contacted with theductal epithelium directly or by expression of a vector comprising agene encoding the factor, in which case the vector can be the same onethat comprises a suicide gene, for example. The factors stimulate apotent, long-lasting, and specific cell immunity, requiring both CD4 andCD8 cells. The immune response is designed to scavenge destroyed ductalepithelial cells by generating autoimuunity towards epithelial cellantigens.

The ductal epithelium is preferably contacted with the agent byintroduction of the agent through the central canal or duct of theexocrine ductal epithelium, such as by ductal cannulation. However, inthe case of the mammary gland, for example, there are 6-9 major ductsthat emanate from the nipple and serially branch into other ducts,terminating in lobulo-alveolar structures (Russo et al. (1990), supra):accordingly, in some circumstances, such as those in which even morelocalized treatment is necessary or desired, for example, by the choiceof anti-cancer agent, it may be preferable to contact the ductalepithelium of the exocrine gland through one of the other ducts orthrough a lobulo-alveolar structure as opposed to the central canal orduct. In this regard, ductal cannulation enables intratumoral injection.

The methods of the present invention can be combined with other methodsof prophylactic and therapeutic treatment in addition to those citedabove, such as methods that target destruction of cancer cells, e.g., bytargeting of cell-surface markers, receptor ligands, e.g., ligands togastrin-releasing peptide-like receptors, tumor-associated antigens,e.g:, the 57 kD cytokeratin or the antigen recognized by the monoclonalantibody GB24, the extracellular matrix glycoprotein tamascin, antisenseoncogenes such as c-fos, homeobox genes that are expressed in cancercells but not normal cells, tumor-infiltrating lymphocytes that expresscytokines, RGD-containing peptides and proteins, which are administeredfollowing surgery, lipophilic drug-containing liposomes to which arecovalently conjugated monoclonal antibodies for targeting to cancercells, low fat diet, moderate physical exercise and hormonal modulation.For prostate cancer, anti-testosterone agents can be used as well as aninhibitor of cell proliferation produced by prostatic stromal cells andC-CAM, an epithelial cell adhesion molecule.

The following examples are presented to illustrate the presentinvention, not to limit its scope. The examples make use of the ratmammary tumor model, which has been deemed an appropriate experimentalmodel for understanding breast cancer in humans (Sukumar et al.,Mutation Res. 333 (1-2): 37-44 (1995); Russo et al., supra). In fact,90-100% of female rats develop mammary tumors in this model when theyare administered the carcinogen NMU at 55 days of age (Sukumar, CancerCells 4: 199-204 (1990)).

EXAMPLES Example 1

This example demonstrates the successful delivery of virus and otheragents into the mammary ductile tree by a single injection through theteat.

ADV/CMV-β-gal (from Dr. William Burns, Johns Hopkins University) is anadenoviral 5 vector constructed with a β-galactosidase gene controlledby a cytomegaloviral promoter. It was delivered into the mammary glandby injection of a viral suspension in 20 μl of 0.2% trypan blue in Trisbuffer through the teat of a rat. The nipple was extruded, and thesphincter removed by excising the nipple. In the rat, the muscleprevents fluid from regurgitating into the breast and had to be excisedin order to visualize the ductal opening and administer the agent.Trypan blue was used as a tracking dye to ensure correct delivery to theductile tree. Injection about 30 days postpartum resulted in the mammaryepithelial tree being clearly visible. Ethyl alcohol (70%) was alsosuccessfully delivered by a single injection through the teat.

Example 2

This example demonstrates that adenovirus can efficiently transducehuman mammary epithelial cells in vitro.

ADV/CMV-β-gal was used to transduce HBL100 mammary epithelial cells invitro. The β-gal enzyme in this construct contains a nuclearlocalization signal and results in dense nuclear staining. HBL100 cells(10²) were plated in 24-well plates, and transduced with virus atvarious doses and stained with X-gal 48 hrs later. Essentially all cellswere infected at a moi=10⁴.

This experiment also has been performed in human mammary tumor cellsMCF-7 (American Type Culture Collection (ATCC), Rockville, Md.), humanmammary epithelial cells MCF-10A (ATCC), and two rat mammary cancer celllines, RBA (from Dr. Leonard Cohen) and 37-2 (from Dr. C. Marcelo Aldaz)with the same results. More efficient adenoviral constructs have beenused, thereby achieving 100% infection at a moi=10³. These experimentsdemonstrate successful infection by and expression of adenoviruscarrying the lacZ indicator gene, which permits staining the cells blueby the expression of the enzyme β-galactosidase.

Example 3

This example demonstrates that infection with an AdHS-tk constructfollowed by GCV treatment effectively kills mammary tumor cells invitro.

RBA and NMU68 are two rat mammary tumor cell lines derived from a DMBA-and a NMU-induced tumor, respectively [DMBA=dimethylbenz[a]anthracene,NMU=N′-nitro N′-methylurea]. Each cell line was plated at a density of5×10² in 48-well plates (1.1 cm) and allowed to settle overnight. Thenext morning, they were transduced with AdHS-tk (Chen et al., PNAS(USA)91: 3054-57 (1994); obtained from S. Woo and E. Aguilar-Cordova, BaylorCollege of Medicine, Houston, Tex.) at titers of 0, 100, 500, and 1000moi, and then, 6 hrs later, GCV (10 μg/ml) was added to the culturemedia. The cells were maintained in the presence of GCV for 3 days andthen counted using trypan blue exclusion as a measure of cell viability.The cell numbers were normalized to the growth of cells in the absenceof GCV. The results are shown in FIG. 1. More than 80% of the cells ofeach cell line were killed at a moi of 10³.

Example 4

This example demonstrates the prophylactic effect of the method of thepresent invention.

The mammary glands (6 on each side) of six virgin 50 day old SpragueDawley rats were injected with AdHS-tk on the left side and trypan blueon the right side or left untreated. One rat remained completelyuntreated with the virus or GCV and served as a positive control forNMU-induced tumorigenesis. On the day of surgery, rats were given anintramuscular injection of 5 μg estradiol valerate and were anesthetizedwith an isofluorane/O₂ mixture. The nipples were cannulated with a 33gauge needle. Twenty μl of AdHS-tk diluted in trypan blue carrier (1 mMMgCl₂, 20 μg/ml polybrene in 10% glycerol, 0.4% trypan blue in saline)at a concentration of 5×10⁷ particles/μl were injected into the duct.Carrier control mammary glands received 20 μl of trypan blue carrieralone. An animal was considered treated when at least three glands weresuccessfully injected with trypan blue and another three glands weresuccessfully injected with AdHS-tk. The remaining glands were leftuntreated. Twelve hrs later, the rats were injected with 125 mg/kg bodyweight GCV twice daily for three days. The rats were then given a secondintramuscular injection of 5 μg estradiol valerate and intraperitonealinjections of 100 μg/kg body weight GCV once daily for three days. Fiveto seven days after GCV treatment, the rats were given an intravenousinjection of NMU dissolved in 0.05% acetic acid (Ash Stevens, CO; 50 mgNMU/kg body weight) and were subsequently monitored for general healthand the appearance of tumors at weekly intervals for 8 months. Theresults were as follows: No. of Total % of Glands Treatment No. ofTumors Glands with Tumor Untreated 5 12 41.7 Control Trypan Blue 5 1729.4 and Ganciclovir No injection; 5 20 25.0 Ganciclovir AdHS-tk and 2*35 5.7 Ganciclovir*The two tumors (size <5 mm) in this group were detected during necropsyat the termination of the experiment 8 months later. The differencebetween tumors appearing in treated versus control glands wassignificant by Chi² analysis (p < 0.01).

The above results show that the method of the present invention inhibitsthe formation of cancer of ductal epithelial origin in this rat model,in which NMU induces the formation of mammary tumors in 90-100% offemale rats of similar age. Surprisingly and unexpectedly, thisprophylactic effect was achieved without extensive destruction of themammary glands. These data demonstrate that selective destruction ofepithelial cells, e.g., key stem cells, can be sufficient to provideprophylactic protection against carcinogen-induced tumor formation inthe ductal epithelium of the mammary gland.

Example 5

This example demonstrates the efficient transfection of mammaryepithelial cells in vivo.

Lytic Vaccinia virus (10⁶ Vaccinia-HA, which also carries lacZ, in 20 μl0.2% trypan blue) was injected into the mammary glands through the teatof 45 day old virgin rats. Contralateral control glands were injectedwith 0.2% trypan blue. The glands were excised after 3 days. Frozenmammary gland sections were stained with X-gal and counterstained witheosin. When Vaccinia-HA was injected via the rat teat, it was able toinfect the epithelial cells. At 3 days post-infection, the X-galstaining was confined primarily to the epithelial cells.

Example 6

This example demonstrates the cytotoxicity of Vaccinia/HA on HBL100cells in vitro.

HBL100 cells (from ATCC, Rockville, Md.) were plated in DME:F12 medium(50% Dulbecco's Modified Eagles Medium: 50% Ham's F12 Supplement)containing 10% fetal bovine serum and 10 μg/ml insulin at a density of5×10⁴ cells/well and incubated at 37° C. overnight. Vaccinia/HA, atconcentrations of 0 moi, 0.1 moi, or 1.0 moi, was added to the culturemedium, and the cells were incubated at 37° C. for at least 3 days. Morethan 90% of the cells were dead within 72 hrs of infection at 0.1 moi.

Example 7

This example shows the death of rat mammary tumor cells in culture byinfection with Vaccinia/HA.

Cells of rat mammary cancer cell line RBA were plated in growth mediumat a density of 5×10⁴ cells/well and incubated at 37° C. overnight.Vaccinia virus engineered to express β-galactosidase and hemagglutiningenes (Vaccinia/HA) was added to the culture medium at concentrations of0 moi, 0.1 moi, or 1.0 moi and incubated at 37° C. for at least 3 days.Up to 90% of the cells were lysed within 72 hours of injection at 1.0moi.

Example 8

This example demonstrates the destruction of mammary epithelium bytransfection with Vaccinia/HA in vivo.

The mammary glands of 45-day old virgin rats were injected through theteat with 1×10⁷ particles of Vaccinia/HA in 20 μl 0.2% trypan blue(tracking dye). Contralateral control glands were injected with 0.2%trypan blue. The glands were excised after 3 days, fixed inchloroform:methanol:acetic acid and stained in iron-hematoxylin.Branching structures of a whole-mounted mammary gland injected withtracking dye alone were visible up to the end buds and alveoli. Alsovisible as brown bodies were the mammary lymph nodes. Examination of awhole-mounted mammary gland of the same rat receiving Vaccinia/HA intrypan blue on the contralateral side revealed that only about 30% ofthe ducts remained. In addition, the lymph nodes were considerablyenlarged, denoting the mounting of an immune response to clear theVaccinia from the vicinity;

All publications, patents, and patent applications cited herein arehereby incorporated by reference to the same extent as if eachindividual document were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

1. An ablation system for an Operator to treat a Patient with Arrhythmiacomprising: an Esophageal Probe for placing in the esophagus of thePatient, said probe comprising a flexible shaft with a proximal end, adistal end, an exterior wall; and a First Location Element; an AblationCatheter including at least one Ablation Element for delivering Energyto Cardiac Tissue, said catheter comprising a flexible shaft with aproximal end, a distal end, an exterior wall, and a Second LocationElement; an Interface Unit for providing energy to the AblationCatheter; and Calculating Means for determining the Distance between theFirst Location Element and the Second Location Element.
 2. The system ofclaim 1 wherein the esophageal probe further comprises at least onetemperature sensor. 3-4. (canceled)
 5. The system of claim 2 wherein aparameter of said system is modified when a sensed temperature reaches athreshold.
 6. The system of claim 5 wherein the system parameter isselected from the group consisting of: a threshold parameter such as anincreased temperature threshold; an alarm parameter such as an alarm onstate; an energy parameter such as a parameter changing energy type ormodifying energy delivery; a sensor parameter such as a parameter whichactivates one or more additional sensors; cooling apparatus parametersuch as a parameter activating a cooling apparatus; and combinationsthereof.
 7. (canceled)
 8. The system of claim 5 wherein the modificationincludes a modification to the energy delivered.
 9. The system of claim8 wherein the modification is selected from the group consisting of:changing the type or types of energy delivered such as from RF tomicrowave energies; changing the intensity of energy delivered includinga stoppage of energy delivery; changing the frequency of energydelivered; changing a pulse width modulation parameter of energydelivered including changing monopolar and bipolar delivery on and offtimes; and combinations thereof.
 10. The system of claim 8 wherein theenergy delivered is delivered heat.
 11. The system of claim 10 whereinthe energy delivered is RF energy
 12. The system of claim 10 whereinmodification is a change from monopolar to bipolar delivery.
 13. Thesystem of claim 12 wherein the temperature threshold is also modified.14. The system of claim 10 wherein modification is a change from bipolarto monopolar delivery.
 15. The system of claim 10 wherein themodification is a change from a phased monopolar-bipolar to bipolardelivery. 16-24. (canceled)
 25. The system of claim 1 wherein either thefirst location element or the second location element comprises atransmitting antenna.
 26. The system of claim 25 wherein the otherlocation element comprises a receiving antenna.
 27. The system of claim25 wherein the other location element is also a transmitting antenna.28-44. (canceled)
 45. The system of claim 1 wherein either the firstlocation element or the second location element is a transmitterselected from the group consisting of: a sound transmitter; anultrasound transmitter and combinations thereof. 46-52. (canceled) 53.The system of claim 45 wherein the transmitter is used to create across-sectional image of the neighboring tissue. 54-55. (canceled) 56.The system of claim 53 further comprising a second catheter includingthe transmitter, said catheter inserted into a lumen of the esophagealprobe. 57-92. (canceled)
 93. The system of claim 1 further comprising afunctional element integrated into one or more of the ablation catheterand the esophageal probe. 94-98. (canceled)
 99. The system of claim 93wherein the functional element is a sensor.
 100. The system of claim 99wherein the sensor is selected from the group consisting of: anelectrical signal sensor such as a cardiac electrode; a temperaturesensor such as a thermocouple; an imaging transducer such as an array ofultrasound crystals; a pressure sensor; a pH sensor; a physiologicsensor such as a blood sensor; a respiratory sensor; an EEG sensor; apulse oximetry sensor; a blood glucose sensor; an impedance sensor; acontact sensor; a strain gauge; an acoustic sensor; and combinationsthereof.
 101. The system of claim 99 wherein the sensor is configured toreceive signals that are indicative of the status of one or more cardiacfunctions of the patient.
 102. The system of claim 101 wherein thereceived signals include electrocardiogram signals.
 103. The system ofclaim 93 wherein the functional element is a sensor configured to mapconductive pathways of the heart of the patient.
 104. The system ofclaim 103 wherein the sensor includes at least one electrode.
 105. Thesystem of claim 104 wherein the at least one electrode is furtherconfigured to deliver energy to tissue.
 106. The system of claim 93wherein the functional element is an acoustic sensor.
 107. The system ofclaim 106 wherein the acoustic sensor provides a signal proportional toa cardiac pulse waveform.
 108. (canceled)
 109. The system of claim 93wherein the functional element is a transmitter configured to emitelectrical energy and/or electrical signals.
 110. The system of claim109 wherein the transmitter is a pacing electrode. 111-117. (canceled)118. The system of claim 109 wherein the transmitter is a defibrillationelectrode having at least one contact.
 119. The system of claim 118further comprising another electrode on the patient's chest in theregion of the sternum, said electrode in electrical communication withthe patient's skin.
 120. The system of claim 119 wherein the esophagealprobe is positioned such that at least one contact of the defibrillationelectrode of the esophageal probe is in the lower third of the patient'sesophagus.
 121. The system of claim 119 further comprising adefibrillation generator for generating electrical pulses transmittedthrough the esophageal wall to defibrillate the patient's heart; whereina first electrical conductor connects to the defibrillation electrode ofthe esophageal probe to said defibrillation generator and a secondelectrical conductor connects the electrode on the patient's chest tosaid defibrillation generator. 122-129. (canceled)
 130. The system ofclaim 93 wherein the functional element is an electrode.
 131. The systemof claim 130 wherein the electrode is a plate or a coil.
 132. The systemof claim 130 wherein the electrode is a sensor and a transmitter.133-196. (canceled)
 197. The system of claim 1 wherein the deliveredenergy is selected from the group consisting of: sound energy such asacoustic energy and ultrasound energy; electromagnetic energy such aselectrical, magnetic, microwave and radiofrequency energies; thermalenergy such as heat and cryogenic energies; chemical energy; lightenergy such as infrared and visible light energies; mechanical energy;radiation; and combinations thereof. 198-201. (canceled)
 202. The systemof claim 1 wherein the interface unit further provides an analysisfunction, said function used to determine an ablation setting selectedfrom the group consisting of: a energy delivery amount; an energydelivery frequency; an energy delivery voltage; an energy deliverycurrent; an energy delivery temperature; an energy delivery rate; anenergy delivery duration; an energy delivery modulation parameter; anenergy threshold; another energy delivery parameter; a temperaturethreshold; an alarm threshold; another alarm parameter; and combinationsthereof.
 203. The system of claim 202 wherein said analysis functionutilizes the distance determined by the calculating means to determineone or more ablation settings. 204-207. (canceled)
 207. The system ofclaim 203 wherein the esophageal probe further comprises a temperaturesensor; and said ablation setting is a temperature threshold correlatingto the temperature threshold.
 208. The system of claim 207 wherein theenergy delivery is modified when the temperature sensor reaches thethreshold temperature.
 209. The system of claim 1 wherein the interfaceunit includes a user interface, said user interface providing means forsetting one or more ablation settings of said system.
 210. The system ofclaim 209 wherein a range of settable values for the one or moreablation settings is modified based on the calculated distance. 211-223.(canceled)
 224. An Ablation System for an Operator to treat a Patientwith Arrhythmia comprising: an Ablation Catheter comprising at least oneAblation Element. said ablation element for delivering Energy to CardiacTissue, said catheter including a proximal end, a distal end, and anexterior wall; an Interface Unit for providing energy to the AblationCatheter; an Esophageal Probe for placing in the esophagus of thePatient, said probe including a proximal end, a distal end, an exteriorwall, a Sensor and a Location Element; and Calculating Means fordetermining the Distance between the Location Element and at least oneof said ablation elements. 225-227. (canceled)
 228. An esophageal probecomprising: an elongate member adapted to be positioned within theesophagus of a patient and having a proximal end, a distal end and anexterior wall; at least one sensor at a location near said distal end;and a visualization transducer at a location near said at least onesensor. 229-236. (canceled)
 237. The esophageal probe of claim 228wherein the sensor is a temperature sensor.
 238. The esophageal probe ofclaim 228 wherein the sensor is selected from the group consisting of:an electrical signal sensor such as a cardiac electrode; a temperaturesensor such as a thermocouple; an imaging transducer such as an array ofultrasound crystals; a pressure sensor; a pH sensor; a physiologicsensor such as a blood sensor, a respiratory sensor; an EEG sensor, apulse oximetry sensor and a blood glucose sensor; an impedance sensor; acontact sensor; a strain gauge; an acoustic sensor; and combinationsthereof. 239-242. (canceled)
 243. A method of treating a patient witharrhythmia comprising: providing an ablation system comprising: anesophageal probe for placing in the esophagus of the Patient, said probecomprising an elongate shaft with a proximal end, a distal end, anexterior wall; and a location element; an interface unit for providingenergy to the Ablation Catheter; an ablation catheter including at leastone ablation element for delivering energy to cardiac tissue, saidcatheter comprising a flexible shaft with a proximal end, a distal end,and an exterior wall, and calculating means for determining the distancebetween the location element and an ablation element; calculating saiddistance between the location element of the esophageal probe and anablation element of the ablation catheter; setting a system parameterbased on said distance delivering energy to said cardiac tissue. 244.The method of claim 243 wherein the ablation catheter further comprisesa location element.
 245. The method of claim 244 wherein said locationelement is an ultrasound transducer.
 246. The method of claim 243wherein the esophageal probe further comprises at least one temperaturesensor.
 247. The method of claim 246 wherein the system parameter set isa temperature threshold.
 248. The method of claim 247 further comprisingthe step of modifying the energy delivered when the temperature sensorreaches the temperature threshold.
 249. The method of claim 248 whereinthe energy modification is a switch from monopolar delivery to bipolardelivery or from bipolar delivery to monopolar delivery.
 250. The methodof claim 248 wherein the energy modification is a decrease in energydelivered. 251-265. (canceled)